Leukemia Cell Line HL-60 Research Articles

Effect of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells on the Proliferation and Apoptosis of Leukemic Cells and Its Mechanism

To investigate the effects of human umbilical cord blood-derived mesenchymal stem cells(HUCMSC) on the leukemic cell line HL-60 and acute lymphoblastic leukemia cell line Jurkat as well as the role of CXCL12/CXCR4. HL-60 cells and Jurkat cells were co-cultured with human umbilical cord blood mesenchymal stem cell (HUCMSC), and the model was treated with G-CSF, AMD3100 and their combination. The cell viability and cell cycle were measured by Cell Counting Kit-8 (CCK-8), the apoptosis and the cell-cycle analysis were assessed by flow cytometry with the Annexin V/PI double staining. The expression of surface CXCR4 protein and total CXCR4 protein of leukemic cells were detected by flow cytometry and Western blot respectively. HUCMSC could decrease the viability of HL-60 cells and Jurkat cells, as well as the percentage of apoptotic cells, they could also increase the number of G0/G1 cells, while G-CSF and AMD3100 could reduce the proliferation of HL-60 cells and Jurkat cells in HUCMSC co-culture model, destructed the anti-apoptotic effect of HUCMSC on HL-60 cells and Jurkat cells, and the combination of 2 drugs resulted in a synergistic effect. The G-CSF could reduce the expression of surface CXCR4 protein and total CXCR4 protein in leukemic cells, while AMD3100 could only decrease the expression of surface CXCR4 protein of leukemia cell membrane, having no effect on the expression of CXCR4 protein in cytoplasm. Human umbilical cord blood mesenchymal stem cells can inhibit the proliferation and apoptosis of acute leukemia cells and increase the number of G0/G1 phase cells in leukemic cells. The AMD3100 can decrease the expression of surface CXCR4 protein in leukemia cells, G-CSF can decrease expression of total CXCR4 protein as well as membrane CXCR4 protein. Both of them can block the CXCL12/CXCR4 signal axis, weakening the relationship between leukemia cells and microenvironment. And on the basic of HUCMSC influenced leukemia cells' growth and proliferation, the cell viability will be weakened, its apoptosis will be promoted, and the percentage of G0/G1 phase cells in leukemia cells will be decreased.

Acute Myelogenous Leukemia Cells Secrete Factors that Stimulate Cellular LDL Uptake via Autocrine and Paracrine Mechanisms

Leukemic cells isolated from most patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) uptake than normal mononuclear blood cells. Little is known, however, about the mechanism behind the elevated LDL uptake. We investigated if AML cells secrete factors that stimulate cellular LDL uptake. Mononuclear blood cells were isolated from peripheral blood from 42 patients with AML at diagnosis. Cellular LDL uptake was determined from the degradation rate of 125I-labelled LDL. Conditioned media from AML cells stimulated the LDL degradation in the leukemic cell lines KG1 and HL60, and in isolated AML cells. The stimulatory effect correlated with the LDL degradation in the AML cells directly after isolation from blood. Conditioned media also autostimulated LDL degradation in the AML cells themselves. Concentrations of IL-6 and IL-8 in AML cell conditioned media correlated with the LDL degradation in AML cells directly after isolation from blood. Addition of R-TNF-α, but not IL-6 or IL-8, stimulated LDL degradation in HL60, KG1, and AML cells. The LDL degradation in AML cells could be inhibited by a LDL receptor blocking antibody. AML cells secrete factors that stimulate LDL uptake in a paracrine and autocrine pattern which open up therapeutic possibilities to inhibit the uptake of LDL by administration of antibodies to these factors.

Inhibition of Ser/Thr phosphatase PPM1D induces neutrophil differentiation in HL-60 cells.

Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy. It has been reported that PPM1D knockout mice exhibit neutrophilia in blood and show a defective immune response. Here, we found that inhibition of PPM1D induced neutrophil differentiation of human promyelocytic leukemia cell line HL-60. The combination of a PPM1D inhibitor and all-trans retinoic acid significantly increased their differentiation efficiency. The PPM1D inhibitor also induced G1 arrest in HL-60 cells. Our results suggest that PPM1D may be a potential therapeutic target for blood cell diseases including leukemia.

Succinamide derivatives of melampomagnolide B and their anti-cancer activities

A series of succinamide derivatives of melampomagnolide B have been synthesized by coupling MMB monosuccinate (2) with various heterocyclic amines to afford compounds 3a–3l. MMB monosuccinate was also reacted with terminal diaminoalkanes to afford dimeric succinamido analogs of MMB (4a–4h). These succinamide analogs of MMB were evaluated for their anti-cancer activity against a panel of sixty human cancer cell lines. Analogs 3d–3i and dimers 4f–4g exhibited promising anti-cancer activity with GI50 values ranging from 0.28 to 33.5µM against most of the cell lines in the panel. The dimeric analogs 4f and 4g were identified as lead compounds with GI50 values in the nanomolar range (GI50=280–980nM) against several cell lines in the panel; i.e. leukemia cell lines CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR; and solid tumor cell lines NCI-H522 (non-small cell lung cancer), SW-620 and HCT-116 (colon cancer), LOX IMVI (melanoma), RXF 393 (renal cancer), and MCF7, BT-549 and MDA-MB-468 (breast cancer). Succinamide analogs 3a, 3c–3l and 4b–4h were also evaluated for their apoptotic activity against M9-ENL1 acute myelogenous leukemia cells; compounds 3h–3j and 4g were equipotent with parthenolide, exhibiting LC50 values in the range 4.1–8.1μM. Molecular docking studies indicate that these molecules interact covalently with the highly conserved Cys-46 residue of the N-terminal lobe (1–109) of human IKKβ to inhibit the NFκB transcription factor complex, resulting in down-regulation of anti-apoptotic genes under NFκB control.

Stereoselective Sequence toward Biologically Active Fused Alkylidenecyclobutanes.

Combining an efficient preparation of cyclobutenylmetal species, high-yielding cross-coupling reactions, and highly diastereoselective [4 + 2]-cycloaddition led to opening a new route toward the synthesis of fused alkylidenecyclobutanes containing up to five consecutive stereocenters. New complex architectures, analogues to protoilludane skeletons, were obtained in a very efficient manner and with a minimum number of steps starting from commercial sources and were tested for their cytotoxicity against leukemia cell lines HL60.

Effects of Atorvastatin on Proliferation and Apoptosis of Leukemia Cell Line HL-60 and Its Mechanism of Signal Pathway

To investigate the effect of atorvastatin on proliferation and apoptosis of leukemia cell line HL-60 and its mechanism of signal pathway. The leukemia HL-60 cells in logarithmic growth phase were seeded in 96 well plates and were treated with 1, 5 and 10 mol/L atorvastatin, then were cultured in the incubator (at 37 °C, 5% CO2) for 12 h, 24 h, 48 h. MTT colorimetric method was used to detect the proliferation leukemia cells, the apoptosis of leukemia cells was detected by flow cytometry; the expresion levels of phosphatidylinositol 3-kinase(PI3K), serine threonine protein kinase(ATK) and mTOR at mRNA and protein levels were detected by RT-PCR and Western blot respectively. The experiments included blank control group, the negative control group and drug-treated group. Atorvastatin could inhibit the proliferation of HL-60 cells. The treatment of HL-60 cells with 10 mol/L atorvastatin for 48 hours showed the strongest inhibition rate (39.78±3.00)% which was statistically significant different from negative control group (t=4.015, P<0.05) and the strongest induction-apoptosis effect on HL-60 cells (43.30±3.92)%, that was statistically significantly different from negative control group (t=3.624, P<0.05). After treatment with atorvastatin for 48 hours, the expression levels of PI3K,ATK and mTOR were decreased, in which the effect of 10 mol/L atorvastatin was the most obvious; The expression levels of PI3K,ATK and mTOR were decreased by (37.04±4.15)%, (53.81±3.25)% and (40.62±2.41) respectively, significantly different from the negative control (t=4.806,3.800,4.313, P<0.05). Atorvastatin may inhibit the proliferation of HL-60 cells and induce apoptosis by inhibiting the PI3K/ATK/mTOR signaling pathway.

Bioactive sterols and sesquiterpenes from the Red Sea soft coral Sinularia terspilli

The ethanolic extract of the Red Sea soft coral Sinularia terspilli was subjected to several chromatographic techniques resulting in the isolation of compounds 1–8 including (five sterols 1–5 and three sesquiterpenes 6–8). The isolated compounds have been identified by 1D and 2D NMR spectral analysis and by comparison with literature data. Different biological activities for the isolated compounds and some synthetic derivatives have been carried out. Compounds 4 and 5 exhibited strong cytotoxic activity against human leukemia cell lines HL60 and K562 with IC50 values ranging from 2.0 to 25 nM. This is the first reported isolation of these metabolites from this soft coral species.

Resistance Studies, in vitro Model, of Myeloid Leukemia Cell Lines HL-60 Against Thymoquinone and Doxorubicin in the Presence of Type I Collagena

Purpose: The prognostic of Acute leukemia is cell drug resistance dependent, which is principal cause of death. The bone marrow microenvironment is directly implicated as source of chemio resistance. Several researchers have studied in vivo and vitro the effect of the bioactive molecules such as the Thymoquinone (TQ) on cancers chemo resistant. The aim of this study is to compare the activities of Thymoquinone to Doxorubicin on presence and on absence of collagen type I, which is the major component of cell extra matrix (CEM). Methods: Cell line HL60 resistance against Doxorubicin and Thymoquinone was tested on presence and on absence Type I collagen at concentration 25, 50 and 100 µg /cm2 TQ and Dox cytototoxicities was evaluated with counting using KOVA Glasstic Slide and phase contrast microscopy. HL-60 cells were seeded at 10 cells/well for 24h in the presence or not of collagen and treated or not with 200nM of Dox or 10 µM of TQ. After incubation, apoptosis was determined using Annex V and Dead Cell Assay kit (Millipore) and Caspase 3/7 Assay kit (Millipore). Results: cell line HL60 proliferation is more resistance against Doxorubicin in presence Type I collagen than Thymoquinone. Conclusion: Collagen induce cell HL60 resistance against Doxorubicin, But not against Thymoquinone. Combination Thymoquinone, bioactive molecule, to Doxorubicin can decrease the drug resistance and improve leukemia prognostic.

Anti-leukemia effects of the novel synthetic 1-benzylindole derivative 21-900 in vitro and in vivo

Cancers are the major cause of death worldwide. Chemotherapy using cytotoxic drugs and targeted therapy is required when surgery is difficult, ineffective, or impossible. We previously synthesized the novel synthetic 1-benzylindole derivative 21-900 and found that it inhibits histone deacetylase (HDAC) activities and tubulin assembly. Here we tested its effects on the human leukaemia cell lines HL-60 and MOLT-4 in vitro and in vivo. We found that its potent cytotoxic effects were mediated through cell cycle arrest at the G2/M phase, which increased the population of sub-G1 cells, leading to apoptosis. Further, tubulin was depolymerized by 21-900 in a manner similar to that of vincristine, leading to disruption of microtubule dynamics and increased levels of the mitotic marker MPM-2. Further, 21-900 increased the expression of cleavage form of poly (ADP-ribose) polymerase (PARP), caspase 3, 7 (cleavage form), and pro-apoptotic protein BAK and decreased the expression of pro-survival BCL-2-family proteins BCL-2, MCL-1, and BID pro-form, leading to the induction of apoptosis. The growth of tumours in nude mice formed by xenografts of HL-60 and MOLT-4 cells was significantly inhibited by 21-900 without causing the mice to lose body weight. These findings indicate that 21-900 may serve as a potent anti-leukaemia drug.

Cytotoxic Activity of Compounds from Styrax obassia

Cancer is a major public health burden in both developed and developing countries. Plant-derived compounds have played an important role in the development of useful anti-cancer agents. The current study was designed to evaluate the cytotoxic activity of chemical compounds from the stem bark of Styrax obassia. Seven known compounds (1-7) were isolated and identified. Compound 2 exhibited cytotoxic activity against the breast cancer cell line MCF-7 with an IC5₅₀ of 27.9 μM, followed by the human cervical cancer cell line Hela with an IC₅₀ of 23.3 μM, and the human promyelocytic leukemia cell line HL-60 with an IC₅₀ of 47.8 RM. Compound 7 exhibited cytotoxicity against Hela cells with an ICso of 16.8 pM, followed by MCF-7 cells with an IC₅₀ of 53.5 μM. This is the first study to investigate the significant anti-tumor properties of isolated compounds from the stem bark of S. obassia.

Effects of TRAM-34 on Proliferation and Invasion of Leukemia Cell Line HL-60

To investigate the effects of KCa3.1 channel inhibitor TRAM-34 on the proliferation and invasion of leukemia cell line HL-60. HL-60 cells at logarithmic growth phase exposed to TRAM-34 at the final concentration of 25, 50, 75 and 100 nmol/L were used as experimental group. The HL-60 cells of control group was cultured in 10% fetal bovine serum-RPMI 1640. The proliferation inhibition rate of TRAM-34 on HL-60 cells was detected by adding MTT solution after 24, 48 and 72 h culture. The cell apoptotic rate and cell cycle distribution of HL-60 cells treated with TRAM-34 were evaluated by flow cytometry with Annexin V-FITC/propidium iodide(PI) double staining or PI single staining. The number of transmembrane cells was detected by Transwell at 24 and 48 h after treatment with TRAM-34. The effect of TRAM-34 on CDK6, P53 and MMP-2 mRNA level was detected by real-time quantitative PCR. Compared with the control group (0 nmol/L), the inhibition rate, apoptosis rate, G0/G1 phase cell proportion and P53 mRNA level all increased, but the percentages of cells in S phase, cell number penetrating the membrane and mRNA levels of CDK6 and MMP-2 in the TRAM-34-treated group decreased (P<0.05) except for 24 h proliferation rate of TRAM-34 at low concentration (25 nmol/L). The effect of TRAM-34 on the above indices was enhanced with the increase of concentration and prolongation of time, and the differences were statistically significant (P<0.05). TRAM-34 can inhibit the proliferation and invasion of HL-60 cells, and can induce cell apoptosis and G0/G1 arrest. The time and concentration of TRAM-34 have effect on the malignant behavior of HL-60 cells.

Isolation and characterisation of cytotoxic compounds from Euphorbia clementei Boiss.

A new phorbol-type diterpene ester, 4,20-dideoxy-4α-phorbol-12β-acetate-13α-isobutyrate, in addition to 11 known compounds were isolated from the latex and roots of Euphorbia clementei Boiss. Structure elucidation was performed by comprehensive 1D and 2D NMR analyses (1H and 13C NMR, COSY, ROESY, HSQC and HMBC experiments), mass spectrometry (HR-ESI-MS) and by comparison with literature data. The inhibitory activity of all isolated compounds was evaluated against promyelocytic leukemia HL60 and human erythromyeloblastoid leukemia K562 cell lines and seven of these compounds exhibited a weak cytotoxicity with IC50 values ranging from 40 to 97 μM.

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

Chemotherapy resistant leukemic stem cells (LSCs) are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP) of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952nM in HL60, 714nM in MOLT4] and relapsed patient blood samples with higher efficacy (85%) than IL2-TRAIL protein (46%). Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

Anti-proliferative effect of eight ethanolic extracts from soft corals on human leukemia cell line HL-60.

In the present study, authors evaluate the anticancer effects of eight crude extracts from 70% ethanol extracts of soft corals collected around Jeju Island, South Korea. According to the cytotoxicity results, extracts are not cytotoxic to Vero cells at the highest tested concentration (100 μg/mL), which indicates that they are not cytotoxic to the normal cells. However, one of the tested extracts, Dendronephthya gigantea (DGI) extract, significantly reduced the viability of human leukemia cell line (HL-60) with IC50 value of 84.43 μg/mL. In addition, Hoechst 33342 cell permeable dye used to visualize the apoptotic nuclei in DGItreated HL-60 cells. These results suggest that DGI has the potential to induce apoptosis in HL-60 cells without altering viability of normal Vero cells. In conclusion, DGI might have the great potential to be developed into an anticancer drug in the future.

Syntheses and anti-cancer activities of glycosylated derivatives of diosgenin

In order to obtain better anti-cancer compounds, nine glycosylated derivatives were designed and synthe-sized using diosgenin as starting material. Their structures were confirmed by 1H NMR, 13C NMR and MS spectra. The anti-cancer activities of intermediate compounds 5a—5i and the target compounds 6a—6i were investigated against human leukemia HEL, K562, HL60 and melanoma WM9 cell lines via MTT method. The bioassay results show that these derivatives possess good inhibitory activities against the four cancer cell lines. Furthermore, these derivatives show better inhibitory activities against K562 than against other cell lines, and most derivatives show better inhibitory activities than the parental material. Moreover, compounds 6a—6i are more active than their inter-mediates 5a—5i when against these cells. The above results demonstrate the effects of glycosylation on 3-OH of di-osgenin and acetylation of the sugar moiety on their antitumor activities.

Identification of the Oncogene SKI As a New Target Gene of the Myeloid Transcription Factor c-MYB in AML

Background:Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB.Methods:In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA).Results:In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB.Conclusion:Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. DisclosuresNo relevant conflicts of interest to declare.

Chemotherapy-Dependent ATP Release from Leukemia Dying Cells Induces Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML.METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells.RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell.CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. DisclosuresMartinelli:Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Transfer of Multidrug Resistance Among Acute Myeloid Leukemia Cells Via Extracellular Vesicles and Their Micrornas Cargo

Resistance of cancer cells to chemotherapy is a major issue in acute myeloid leukemia (AML) and is usually due to a clonal selection of resistant leukemic cells. Recently, a horizontal transfer of chemoresistance among tumor cells has been reported via extracellular vesicles (EVs). EVs are vesicles ranging from 0.03 to 1µm generated by almost all cell types. They carry cellular components such as nucleic acids, membrane and cytosolic proteins. By interacting with cells and by transferring their content, these vesicles could modify target cells’ phenotype. Because of their presence in biological fluids, a lot of studies have tried to use EVs as biomarkers.The aim of this research is to investigate the role of EVs in chemoresistance in AML. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was compared to its multiresistant strain: HL60/AR. The latter overexpresses multidrug resistance-associated protein 1 (MRP1), an efflux pump conferring resistance to anthracyclines. The transfer of this resistance between the two strains is obtained by treating HL60 with EVs generated by HL60/AR.The production of EVs by HL60/AR was confirmed by transmission electron microscopy. EVs from around 100 to 800 nm were highlighted. EVs from HL60/AR were then isolated by ultracentrifugation and their interaction with HL60 was investigated by labeling EVs with a fluorescent lipid membrane linker. The transmission of this fluorescence to HL60 was monitored by flow cytometry and demonstrated a binding or an incorporation of EVs.In order to check if this interaction has an impact on HL60 resistance to daunorubicin, HL60 were treated with EVs, once for 20 hours or four consecutive times for 48 hours each. The resistance to daunorubicin of EVs-treated HL60 was then assessed by a MTT cytotoxicity assay and revealed that EVs increase survival of cells to daunorubicin. The resistant profile between the cells treated once or four times with EVs were somewhat different and highlighted a potential implication of nucleic acids cargo in this resistance transfer. Indeed, nucleic acids transferred by EVs would need more than 20 hours to impact target cells.Afterwards, we investigated the changes in phenotype that could be responsible for this increased resistance of EVs-treated cells. First, the expression of MRP1 was evaluated by a flow cytometry drug retention assay based on the fluorescence exhibited by daunorubicin and consisting in analysis by flow cytometry of the daunorubicin retention of cells with or without the presence of a specific MRP1 inhibitor. This experiment emphasized an expression of MRP1 after EVs treatment. Oxidative stress also plays an important role in daunorubicin cytotoxicity. Thus reactive oxygen species (ROS) generation assays were performed on EVs-treated cells. These cells showed a decreased production of daunorubicin-induced ROS.We finally analyzed the microRNAs content of isolated EVs. This was performed by TaqMan® Human MicroRNA Array on EVs from HL60/AR but also on EVs generated by HL60. This experiment aimed at highlighting the microRNAs cargo potentially involved in this chemoresistance transfer but also at identifying microRNAs differentially expressed between the two strains to serve as biomarker of chemoresistance in vivo. Out of the 380 microRNAs tested, 32 presented a differential expression between HL60 and HL60/AR. Among them, 5 microRNAs presented a more than 8-fold change between the two strains: miR-19b, miR-20a, miR-488, miR-519a and miR-523. miR-519a was the only microRNA more expressed in EVs produced by HL60, compared to EVs from HL60/AR.In conclusion, this study demonstrates the ability of resistant leukemic cells to transfer, at least partially, their chemoresistance via EVs. This could be due to a direct transfer of MRP1 transported by EVs but also to a nucleic acid transfer. In this context, some microRNAs have been highlighted for their high differential expression in EVs between sensitive and chemoresistant cells. These results bring out a potential clinical use of these circulating microRNAs as chemoresistance biomarkers in AML. DisclosuresNo relevant conflicts of interest to declare.

Critical roles of matrix metalloproteinases secreted by leukemic cells in the pathogenesis of central nervous system leukemia

目的观察白血病细胞分泌的基质金属蛋白酶（MMP）-2和MMP-9对脑微血管内皮细胞（BMVEC）紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障（BBB）通透性的影响，探讨MMP-2和MMP-9在中枢神经系统白血病（CNSL）发病机制中的作用。方法①实时定量PCR检测SHI-1、HL-60、U937细胞MMP-2、MMP-9基因的转录水平；明胶酶谱法检测细胞培养上清中MMP-2和MMP-9蛋白表达；体外穿膜实验观察各白血病细胞株的侵袭能力。②将原代人BMVEC接种于铺有Matrigel胶和纤维黏连蛋白包被的Transwell小室系统中，建立体外BBB模型。将蛋白酶抑制剂GM6001处理或未处理的SHI-1、HL-60、U937细胞或MMP-2/MMP-9基因沉默的SHI-1细胞接种于BBB模型的Transwell小室上层与BMVEC共培养，倒置相差显微镜观察BMVEC的形态变化，激光共聚焦显微镜观察紧密连接蛋白ZO-1、claudin-5和occludin的表达，计算白血病细胞的穿膜率。结果①SHI-1细胞表达较高转录水平的MMP-2和MMP-9及酶活性，且侵袭能力强于HL-60、U937细胞（P< 0.01）。②与HL-60、SHI-1和U937细胞共培养后，融合致密的BMVEC之间出现间隙、细胞呈单个生长，紧密连接蛋白ZO-1、claudin-5和occludin的表达明显下调，各白血病细胞均不同程度地穿过体外BBB进入Transwell小室下层。其中SHI-1细胞对BMVEC的形态改变及3种紧密连接蛋白的下调最为明显，穿膜率最高。GM6001明显抑制白血病细胞分泌MMP-2和MMP-9，使BMVEC的形态有所恢复，同时上调ZO-1、claudin-5和occludin的表达，降低了BBB的通透性。③用siRNA分别沉默MMP-2和MMP-9基因后，SHI-1细胞分泌MMP-2和MMP-9被抑制，SHI-1细胞穿膜率较沉默前分别下降43.64％和57.30%（P<0.01），ZO-1、claudin-5和occludin表达上调。结论白血病细胞株分泌的MMP-2和MMP-9能通过降解BMVEC紧密连接蛋白ZO-1、claudin-5和occludin而破坏BBB。

Small-Molecule Disruption of the Myb/p300 Cooperation Targets Acute Myeloid Leukemia Cells.

The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacologic inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line, we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the coactivator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphthoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb. Mol Cancer Ther; 15(12); 2905-15. ©2016 AACR.